Poly(ADP-ribose) polymerase (“PARP”) is an enzyme located in the nuclei of cells of various organs, including muscle, heart and brain cells. PARP plays a physiological role in the repair of strand breaks in DNA. Once activated by damaged DNA fragments, e.g., after exposure to chemotherapy, ionizing radiation, oxygen free radicals, or nitric oxide (NO), PARP catalyzes the transfer of ADP-ribose units from nicotinamide adenine dinucleotide (NAD+) to nuclear acceptor proteins, and is responsible for the formation of protein-bound linear and branched homo-ADP-ribose polymers. The PARP's activation results in the attachment of up to 100 ADP-ribose units to a variety of nuclear proteins, including histones, topoisomerases, DNA and RNA polymerases, DNA ligases, Ca2+- and Mg2+-dependent endonucleases and PARP itself. While the exact range of functions of PARP has not been fully established, this enzyme is thought to play a role in enhancing DNA repair and maintaining DNA integrity.
During major cellular stresses, however, the extensive activation of PARP can rapidly lead to cell damage or death through depletion of energy stores. Four molecules of ATP are consumed for every molecule of NAD (the source of ADP-ribose) regenerated. Thus, NAD, the substrate of PARP, is depleted by massive PARP activation and, in the efforts to re-synthesize NAD, ATP may also be depleted.
It has been reported that PARP activation plays a key role in both NMDA- and NO-induced neurotoxicity, as shown by the use of PARP inhibitors to prevent such toxicity in cortical cultures in proportion to their potencies as inhibitors of this enzyme (ZHANG et al., Science, vol. 263, p: 687-89, 1994); and in hippocampal slices (WALLIS et al., NeuroReport, vol. 5(3), p: 245-48, 1993). The potential role of PARP inhibitors in treating neurodegenerative diseases and head trauma has thus been known. Research, however, continues to pinpoint the exact mechanisms of their salutary effect in cerebral ischemia, (ENDRES et al., J. Cereb. Blood Flow Metabol., vol. 17, p: 1143-51, 1997) and in traumatic brain injury (WALLIS et al., Brain Res., vol. 710, p: 169-77, 1996).
The PARP inhibitors are additionally useful for treating cardiovascular diseases. Ischemia, a deficiency of oxygen and glucose in a part of the body, can be caused by an obstruction in the blood vessel supplying that area or a massive hemorrhage. Two severe forms, heart attack and stroke, are major killers in the developed world. Cell death results directly and also occurs when the deprived area is reperfused. It has been demonstrated that single injections of PARP inhibitors have reduced the infarct size caused by ischemia and reperfusion of the heart or skeletal muscle in rabbits. In these studies, a single injection of the PARP inhibitor, 3-amino-benzamide (10 mg/kg), either one minute before occlusion or one minute before reperfusion, caused similar reductions in infarct size in the heart (32-42%). Another PARP inhibitor, 1,5-dihydroxyisoquinoline (1 mg/kg), reduced infarct size by a comparable degree (38-48%; THIEMERMANN et al., Proc. Natl. Acad. Sci. USA, vol. 94, p: 679-83, 1997). This finding has suggested that PARP inhibitors might be able to salvage previously ischemic heart or skeletal muscle tissue. Presently, PARP inhibitors are being developed to treat ischemia/reperfusion injuries (ZHANG, The Prospect for Improved Medicines, Ashley Publications Ltd, 1999).
PARP activation has also been shown to provide an index of damage following neurotoxic insults by glutamate (via NMDA receptor stimulation), reactive oxygen intermediates, amyloid .beta.-protein, n-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite N-methyl-4-phenylpyridine (MPP+), which participate in pathological conditions such as stroke, Alzheimer's disease and Parkinson's disease (ZHANG et al., J. Neurochem., vol. 65(3), p: 1411-14, 1995). Other studies have continued to explore the role of PARP activation in cerebellar granule cells in vitro and in MPTP neurotoxicity (COSI et al., Ann. N. Y. Acad. Sci., vol. 825, p: 366-79, 1997; COSI et al., Brain Res., vol. 729, p: 264-69, 1996).
Neural damage following stroke and other neurodegenerative processes is thought to result from a massive release of the excitatory neurotransmitter glutamate, which acts upon the N-methyl-D-aspartate (NMDA) receptors and other subtype receptors. Glutamate serves as the predominate excitatory neurotransmitter in the central nervous system (CNS). Neurons release glutamate in great quantities when they are deprived of oxygen, as may occur during an ischemic brain insult such as a stroke or heart attack. This excess release of glutamate in turn causes over-stimulation (excitotoxicity) of N-methyl-D-aspartate (NMDA), AMPA, Kainate and MGR receptors. When glutamate binds to these receptors, ion channels in the receptors open, permitting flows of ions across their cell membranes, e.g., Ca2+ and Na+ into the cells and K+ out of the cells. These flows of ions, especially the influx of Ca2+, cause overstimulation of the neurons. The over-stimulated neurons secrete more glutamate, creating a feedback loop or domino effect which ultimately results in cell damage or death via the production of proteases, lipases and free radicals. Excessive activation of glutamate receptors has been implicated in various neurological diseases and conditions including epilepsy, stroke, Alzheimer's disease, Parkinson's disease, Amyotrophic Lateral Sclerosis (ALS), Huntington's disease, schizophrenia, chronic pain, ischemia and neuronal loss following hypoxia, hypoglycemia, ischemia, trauma, and nervous insult. Recent studies have also advanced a glutamatergic basis for compulsive disorders, particularly drug dependence. Evidence includes findings in many animal species, as well as, in cerebral cortical cultures treated with glutamate or NMDA, that glutamate receptor antagonists block neural damage following vascular stroke (DAWSON et al., H. Hunt Batjer ed., p: 319-25, 1997). Attempts to prevent excitotoxicity by blocking NMDA, AMPA, Kainate and MGR receptors have proven difficult because each receptor has multiple sites to which glutamate may bind. Many of the compositions that are effective in blocking the receptors are also toxic to animals. As such, there is no known effective treatment for glutamate abnormalities.
The stimulation of NMDA receptors, in turn, activates the enzyme neuronal nitric oxide synthase (NNOS), which causes the formation of nitric oxide (NO), which more directly mediates neurotoxicity. Protection against NMDA neurotoxicity has occurred following treatment with NOS inhibitors (DAWSON et al., Proc. Natl. Acad. Sci. USA, vol. 88, p: 6368-71, 1991; DAWSON et al., J. Neurosci., vol. 13(6), p: 2651-61, 1993). Protection against NMDA neurotoxicity can also occur in cortical cultures from mice with targeted disruption of NNOS (DAWSON et al., J. Neurosci., vol. 16(8), p: 2479-87, 1996).
It is known that neural damage following vascular stroke is markedly diminished in animals treated with NOS inhibitors or in mice with NNOS gene disruption (IADECOLA, Trends Neurosci., vol. 20(3), p: 132-39, 1997; HUANG et al., Science, vol. 265, p: 1883-85, 1994; BECKMAN et al., Biochem. Soc. Trans., vol. 21, p: 330-34, 1993). Either NO or peroxynitrite can cause DNA damage, which activates PARP. Further support for this is provided in SZABO et al. (Proc. Natl. Acad. Sci. USA, vol. 93, p: 1753-58, 1996).
It is also known that PARP inhibitors affect DNA repair generally. CRISTOVAO et al. (Terato., Carcino., and Muta., vol. 16, p: 219-27, 1996) discusses the effect of hydrogen peroxide and .gamma.-radiation on DNA strand breaks in the presence of and in the absence of 3-aminobenzamide, a potent inhibitor of PARP. CRISTOVAO et al. observed a PARP-dependent recovery of DNA strand breaks in leukocytes treated with hydrogen peroxide.
Evidence also exists that PARP inhibitors are useful for treating inflammatory conditions such as inflammatory bowel disorders (SOUTHAN et al., Br. J. Pharm., vol. 117, p: 619-32, 1996; SZABO et al., J. Biol. Chem., vol. 272, p: 9030-36, 1997) or arthritis (SZABO et al., Portland Press Proc., vol. 15, p: 280-281, 1998; SZABO, Eur. J. Biochem., vol. 350(1), p: 1-19, 1998; SZABO et al., Proc. Natl. Acad. Sci. USA, vol. 95(7), p: 3667-72, 1998; SZABO et al., Proc. Natl. Acad. Sci. USA, vol. 93, p: 1753-58, 1996; BAUER et al., Intl. J. Oncol., vol. 8, p: 239-52, 1996; HUGHES et al., J. Immuno., vol. 153, p: 3319-25, 1994). Thus, SALZMAN et al. (Japanese J. Pharm., vol. 75 (Supp. I), p: 15, 1997) shows that 3-aminobenzamide, a specific inhibitor of PARP activity, reduced the inflammatory response and restored the morphology and the energetic status of the distal colon in rats suffering from colitis induced by intraluminal administration of the hapten trinitrobenzene sulfonic acid in 50% ethanol. As another example, SZABO et al. (Japanese J. Pharm., vol. 75 (Supp. I), p: 102, 1997) discusses the ability of PARP inhibitors to prevent or treat collagen-induced arthritis.
Further, PARP inhibitors appear to be useful for treating diabetes and have been studied at the clinical level to prevent development of insulin-dependent diabetes mellitus in susceptible individuals (SALDEEN et al., Mol. Cellular Endocrinol., vol. 139, p: 99-107, 1998). In models of Type I diabetes induced by toxins such as streptozocin and alloxan that destroy pancreatic islet cells, it has been shown that knock-out mice lacking PARP are resistant to cell destruction and diabetes development (PIEPER et al., Trends Pharmacolog. Sci., vol. 20, p: 171-181, 1999; BURKART et al., Nature Medicine, vol. 5, p: 314-319, 1999). Administration of nicotinamide, a weak PARP inhibitor and a free-radical scavenger, prevents development of diabetes in a spontaneous autoimmune diabetes model, the non-obese, diabetic mouse (PIEPER et al., 1999, aforementioned). Hence, potent and specific PARP inhibitors may be useful as diabetes-prevention therapeutics.
Further still, PARP inhibitors have been shown to be useful for treating endotoxic shock or septic shock (ZINGARELLI et al., Shock, vol. 5, p: 258-64, 1996; CUZZOCREA, Brit. J. Pharm., vol. 122, p: 493-503, 1997). ZINGARELLI et al. suggests that inhibition of the DNA repair cycle triggered by poly(ADP ribose) synthetase has protective effects against vascular failure in endotoxic shock. ZINGARELLI et al. found that nicotinamide protects against delayed, NO-mediated vascular failure in endotoxic shock. ZINGARELLI et al. also found that the actions of nicotinamide may be related to inhibition of the NO-mediated activation of the energy-consuming DNA repair cycle, triggered by poly(ADP ribose) synthetase.
Yet another known use for PARP inhibitors is treating cancer. In fact, the PARP's activity can contribute to the resistance that often develops to various types of cancer therapies, because this cellular ADP-ribose transfer process is associated with the repair of DNA strand breakage in response to DNA damage caused by radiotherapy or chemotherapy. Consequently, inhibition of PARP may retard intracellular DNA repair and enhance the antitumor effects of cancer therapy. Indeed, in vitro and in vivo data show that many PARP inhibitors potentiate the effects of ionizing radiation (U.S. Pat. Nos. 5,032,617; 5,215,738; 5,041,653; 5,177,075) or cytotoxic drugs such as alkylating agents (WELTIN et al., Oncol. Res., vol. 6(9), p: 399-403, 1994). Thus, inhibitors of the PARP enzyme are useful as adjunct cancer chemotherapeutics.
Still another use for PARP inhibitors is the treatment of peripheral nerve injuries, and the resultant pathological pain syndrome known as neuropathic pain, such as that induced by chronic constriction injury (CCI) of the common sciatic nerve and in which transsynaptic alteration of spinal cord dorsal horn characterized by hyperchromatosis of cytoplasm and nucleoplasm (so-called “dark” neurons) occurs (MAO et al., Pain, vol. 72, p: 355-366, 1997).
PARP inhibitors have also been used to extend the lifespan and proliferative capacity of cells including treatment of diseases such as skin aging (U.S. Pat. No. 5,589,483), Alzheimer's disease, atherosclerosis, osteoarthritis, osteoporosis, muscular dystrophy, degenerative diseases of skeletal muscle involving replicative senescence, age-related macular degeneration, immune senescence, AIDS, and other immune senescence diseases; and to alter gene expression of senescent cells.
Large numbers of PARP inhibitors have been described. For example, BANASIK et al. (J. Biol. Chem., vol. 267(3), p: 1569-75, 1992) examined the PARP-inhibiting activity of over one hundred compounds, the most potent of which were 4-amino-1,8-naphthalimide, 6(5H)-phenanthridone, 2-nitro-6(5H)-phenanthridone, and 1,5-dihydroxyisoquinoline. GRIFFIN et al. reported the PARP-inhibiting activity for certain benzamide compounds (Anti-Cancer Drug Design, vol. 10, p: 507-514, 1995; U.S. Pat. No. 5,756,510), benzimidazole compounds (WO 97/04771), and quinalozinone compounds (WO 98/33802). SUTO et al. reported PARP inhibition by certain dihydroisoquinoline compounds (Anti-Cancer Drug Design, vol. 7, p: 107-117, 1991). GRIFFIN et al. have reported other PARP inhibitors of the quinazoline class (J. Med. Chem., vol. 41, p: 5247-5256, 1998). Finally, WO 99/11622, WO 99/11623, WO 99/11624, WO 99/11628, WO 99/11644, WO 99/11645, and WO 99/11649 also describe various PARP-inhibiting compounds.
However, the approach of using these PARP inhibitors in the ways discussed above has been limited in effect. For example, side effects have been observed with some of the best-known PARP inhibitors (MILAM et al., Science, vol. 223, p: 589-91, 1984). Specifically, the PARP inhibitors 3-aminobenzamide and benzamide not only inhibited the action of PARP but also were shown to affect cell viability, glucose metabolism, and DNA synthesis. Thus, it was concluded that the usefulness of these PARP inhibitors may be severely restricted by the difficulty of finding a dose that will inhibit the enzyme without producing additional metabolic effects.
Accordingly, there remains a need for compounds that inhibit more specifically PARP activity, compositions containing those compounds, and methods utilizing those compounds, wherein the compounds produce more potent and reliable effects with fewer side effects, with respect to inhibiting PARP activity and treating the diseases and conditions discussed herein.
Macro-H2A1 and macroH2A2 histones are particularly enigmatic histone variants having an N-terminal region with high sequence homology to H2A, and also containing an extensive non histone C-terminal tail that comprises nearly two third of the protein (25 kDa). The human genome contains two genes that code for macroH2A histones. The MACROH2A1 gene encodes two subtypes MACROH2A1.1 and MACROH2A1.2 produced by alternative splicing. A second gene codes for MACROH2A2 (CHADWICK and WILLARD, Human. Mol. Genet., vol. 10, p: 1101-1113, 2001). These proteins appear to be enriched in heterochromatin such as the inactive X chromosome (Xi) in female mammals (COSTANZI and PEHRSON, Nature, vol. 393, p: 599-601, 1998), and discrete heterochromatic loci in senescent and quiescent cells (ZHANG et al., Dev. Cell, vol. 8, p: 19-30, 2005; GRIGORYEV et al., J. Cell. Sci., vol. 117, p: 6153-6162, 2004). MacroH2A is highly localized in female cells as a distinct nuclear body, referred to as a macro chromatin body (MCB), which is coincident with the Xi and the Barr body (COSTANZI and PEHRSON, aforementioned, 1998). The C-terminal region of macroH2A contains a domain termed “macro domain”, which is found alone or in multiple copies in a number of otherwise unrelated proteins (PEHRSON and FUJI, Nuc. Acids Res., vol. 26, p: 2837-2849, 1998), and which is critical for macroH2A MCB formation (CHADWICK et al., Nuc. Acids Res., vol. 29(13), p: 2699-2705, 2001). It has been suggested that this macro domain defines a superfamily of phosphoesterases that act on ADP ribose derivatives (ALLEN et al., J. Mol. Biol., vol. 330, p: 503-511, 2003). Recently, KUSTATSCHER et al. (Nat. Struct. Mol. Biol., vol. 12(7), p: 624-5, 2005) shows that macroH2A1.1 binds to monomeric ADP-ribose and to O-acetyl-ADP-ribose (a NAD metabolite). The authors identify Phe348, Asp203, Gly224 and Gly314 as critical residues for O-acetyl-ADP-ribose binding. Nevertheless, the specific macroH2A function is still unknown.